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Aquatic Toxicology

BICARBONATE TOXICITY TO CERIODAPHNIA DUBIA AND THE FRESHWATER SHRIMP

PARATYA AUSTRALIENSIS AND ITS INFLUENCE ON ZINC TOXICITY

CAROLINA LOPEZ VERA, ROSS V. HYNE, RON PATRA, SUNDERAM RAMASAMY, FLEUR PABLO, MORENO JULLI,

AND BEN J. KEFFORD

Environ Toxicol Chem., Accepted Article • DOI: 10.1002/etc.2545 Accepted Article "Accepted Articles" are peer-reviewed, accepted manuscripts that have not been edited, formatted, or in any way altered by the authors since acceptance. They are citable by the Digital Object Identifier (DOI). After the manuscript is edited and formatted, it will be removed from the “Accepted Articles” Web site and published as an Early View article. Note that editing may introduce changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. SETAC cannot be held responsible for errors or consequences arising from the use of information contained in these manuscripts.

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t Aquatic Toxicology Environmental Toxicology and Chemistry DOI 10.1002/etc.2545

BICARBONATE TOXICITY TO CERIODAPHNIA DUBIA AND THE FRESHWATER SHRIMP

PARATYA AUSTRALIENSIS AND ITS INFLUENCE ON ZINC TOXICITY

CAROLINA LOPEZ VERA,† ROSS V. HYNE,*‡ RON PATRA,‡ SUNDERAM RAMASAMY,‡ FLEUR PABLO,‡ MORENO

JULLI,‡ AND BEN J. KEFFORD†

† Centre for Environmental Sustainability, University of Technology Sydney, Broadway, New South Wales,

AUSTRALIA

‡ Centre for Ecotoxicology, Office of Environment and Heritage, Lidcombe, New South Wales, AUSTRALIA

Running title: Toxicity of bicarbonate and its influence on zinc toxicity

*Address correspondence to Ross.Hyne@environment.nsw.gov.au.

© 2014 SETAC Submitted 20 September 2013; Returned for Revisions 28 January 2014; Accepted 30 January 2014

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Abstract: Bicarbonate is often a major ionic constituent associated with produced waters from methane gas

extraction and coal mining but few studies have determined its specific toxicity. Currently the environmental risk

of bicarbonate anion in water discharges is assessed based on the toxicity of sodium chloride or artificial sea water

and is regulated via electrical conductivity. Increased NaHCO3 added to Ceriodaphnia dubia in synthetic or natural

water gave similar 48-h EC10 values of 1750 ± 125 (mean ± SE) and 1670 ± 180 mg NaHCO3/L, respectively.

Bicarbonate was toxic to C. dubia in both waters with conductivities above 1900 µS/cm. In contrast, when

conductivity was elevated with NaCl, toxicity to C. dubia was only observed above 2800 µS/cm. Bicarbonate also

impaired C. dubia reproduction with an EC10 of 340 mg NaHCO3/L. Major ion composition also influenced Zn

bioavailability, a common co-occurring metal contaminant in coal mine waters with sub-lethal concentrations of

NaHCO3 and elevated pH increasing Zn toxicity. Higher pH was the dominant parameter determining a 10-fold

increase in the 48-h EC50 for Zn toxicity to C. dubia at pH 8.6 of 34 µg Zn/L (95% CL = 32–37) compared to

the Zn toxicity at approximately circumneutral pH. Exposure of the freshwater shrimp Paratya australiensis

(Atyidae) in natural water to increasing bicarbonate gave a mean 10-d LC10 of 850 ± 115 mg NaHCO3/L,

associated with a mean conductivity EC10 of 1145 µS/cm, which is considerably lower than toxicity of NaCl and

artificial sea water to this species reported elsewhere. Since toxicity was influenced by salt composition, specific

ions should be regulated rather than conductivity alone in mine waste water discharges.

Keywords: Bicarbonate, Zinc, Alkalinity, Cladoceran, Major ions, Salinity

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INTRODUCTION

Coal bed methane extraction has increased world-wide over the past two decades since beginning in the

United States in the 1970s. The waters produced from the extraction process of the underlying coal beds as well as

in discharge waters from coal mining are high in salinity. High levels of salinity are known to affect biota [1–3],

especially freshwater organisms and ecosystems. Bicarbonate is one of the major ionic constituents associated with

discharge waters from coal resource extraction along with metals and hydrocarbons naturally found within the coal

bed [4,5]. Studies examining the toxicity of major ions to Ceriodaphnia dubia and Pimephales promales [6,7]

suggested NaHCO3 as one of the salts most likely to be contributing to the toxicity of alkaline produced waters;

however little is known about the toxicity of bicarbonate to aquatic life and its ecological effects.

The ecological safety of disposal of saline coal mine waters cannot yet be determined. Existing salinity

sensitivity data are likely of limited relevance for predicting effects of discharge of most saline effluents from

resource extraction because greater than 90% of toxicological data for salinity sensitivity of freshwater biota uses

artificial sea water or sodium chloride (NaCl) as the salt source. Sea water is approximately 85% NaCl and in

Australia most agricultural related salinity has ionic proportions similar to those of sea water [8]. Nonetheless,

saline effluents from hydrocarbon extractions are not similar to sea water and are very variable. They often have

higher proportions of bicarbonate (HCO3¯), sulphate (SO4

2¯) and boron in addition to metal and organic

contaminants [4,5,9].

The proportions of ions in saline waters have major effects on its toxicity [6] and it has been recently

suggested that ionic proportions of HCO3¯ SO4

2¯, Ca2+ and Mg2+ [9] or HCO3¯ alone [10] may be more important

in determining ecological effects than the total salinity. Yet the water quality guidelines in Australia, Canada or the

United States do not give values to protect aquatic life for anions such as HCO3¯ and SO4

2¯. Following the United

States Environmental Protection Agency (USEPA) procedure for calculating water quality criteria, Farag and

Harper [10] proposed a criterion maximum concentration or an acute guideline value of 317 mg/L HCO3– and a

criterion continuous concentration or chronic guideline value of 290 mg/L HCO3– for the protection of aquatic life.

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t In addition, other studies have shown that bicarbonate alkalinity can modulate the toxicity of a number of metal

ions such as Cu2+ and Pb2+, which are common contaminants in coal mine discharge waters, to C. dubia [11,12].

Currently discharge waters from coal mines in Australia are commonly only regulated by physico-chemical

parameters such as water salinity limits [http://www.epa.nsw.gov.au/prpoeo/index.htm]. There is a need to develop

guidelines to improve the management of the major anions such as bicarbonate which can be a major contributor to

the toxicity of these waters. Freshwater crustaceans are sensitive species to changes in water quality parameters

such as alkalinity and hardness that can influence the toxicity of chloride, SO42¯ as well as metal ions [13–16]. This

current study determined bicarbonate toxicity to two freshwater crustacean species in synthetic or natural water.

The relationship of increasing bicarbonate or alkaline pH to the acute toxicity of zinc, a common contaminant in

coal mine discharge water, to the cladoceran Ceriodaphnia dubia in synthetic water was also examined.

MATERIALS AND METHODS

Cladoceran culture methods

The test organisms were < 24-h old neonates of the cladoceran, Ceriodaphnia dubia sensu stricto. This

species is taxonomically similar to C. dubia Richard, 1894 the type species (D. Berner, pers comm.). The cultures

have been maintained in the Centre for Ecotoxicology (NSW) laboratories based on the procedures previously

described [11] using dechlorinated Sydney tap water. Residual chlorine was measured and removed by the addition

of sodium thiosulphate. This thiosulphate-treated Sydney tap water was supplemented with 5% Perrier mineral

water and filtered seawater to a conductivity of 500 µS/cm at 25C 1. This water was used as the cladoceran

culture and test water (alkalinity 50–69 mg CaCO3/L and hardness 80–100 mg CaCO3/L). Ten neonates from each

cladoceran culture were assessed weekly by determining the number of young produced in three broods compared

against a threshold level (mean of 16 young per cladoceran) to ensure that the cladoceran cultures were in a healthy

state. The cladocerans were fed with two species of green algae, Ankistrodesmus sp. and Pseudokirchneriella

subcapitata (50,000 cells/mL of each species) as well as Yeast, Cerophyl® and Trout Chow (YCT) food

supplement (0.1 mL YCT/animal) [17].

The YCT supplement was prepared based on the USEPA protocol [17] except that a dried powdered blend of

barley and wheat grass (Lifestream International Ltd) was used instead of Cerophyl and a 3:2 mixture of fish food

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t flakes (Sera Vipan) and Sera micron (Sera) used instead of Trout Chow.

Synthetic water

The control water for toxicity testing was USEPA synthetic soft water (hardness 44 mg CaCO3/L; alkalinity

30 mg CaCO3/L; conductivity 160 to 180 µS/cm) [17] made from the following analytical reagent grade chemicals

(BDH-Merck); sodium bicarbonate (NaHCO3), calcium sulfate, magnesium sulfate and potassium chloride added

to purified Milli-Q water (Millipore Australia Pty Ltd). For toxicity tests involving varying concentrations of

NaHCO3, the USEPA synthetic soft water was modified by the addition of 5 mM of the buffer 3-

morpholinopropanesulfonic acid (MOPS) together with 4 mM of MOPS sodium salt (Sigma-Aldrich) to pH 7.1. To

examine the effect of high pH alone with no added NaHCO3 on Zn toxicity, 4 mM Piperazine-N,N'-bis (4-

butanesulfonic acid) (PIPBS) (GFS Chemicals) was added to the modified USEPA soft water and the pH adjusted

to 8.1 or 8.6 with NaOH. These buffers were selected on the basis to reduce the rise in pH associated with the

added NaHCO3 to increase the alkalinity or to determine Zn toxicity at higher pH to C. dubia with no added

NaHCO3 present.

Cladoceran acute toxicity test

The cladoceran acute toxicity test method used was based on the USEPA acute toxicity test protocol [17].

Neonates (<24-h old) of C. dubia were exposed for 48-h to waters with adjusted physicochemical parameters. Five

neonates were added to each of four replicates to give a total of 20 per treatment. Each experiment was repeated a

number of times (typically four times) using different batches of neonates. The test vessels were 100 mL glass

beakers containing 50 mL of test water, which was not renewed during the test. At least five salt or Zn

concentrations and a control were used. All tests were conducted using static procedures with a 16:8 h light: dark

photoperiod at 25°C ± 1.0°C and a light intensity of 400–600 lux at the surface of the test solution. The

cladocerans tested in the natural water were not fed, but the cladocerans tested in the synthetic water were fed with

a half-dose of food at the beginning of the test. Observations of the number of immobilized animals were made at

24 and 48-h. Immobilisation is defined as the cessation of all visible signs of movement or activity, including

second antennae and abdominal legs when viewed under a10x magnification of a dissecting microscope.

Physicochemical parameters including pH, temperature, conductivity, dissolved oxygen and concentrations of total

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t zinc from one replicate of each treatment were measured at 0-h and 48-h. Measurement of pH, conductivity,

temperature and dissolved oxygen measurements were made with calibrated probes from WTW (Weilheim).

The control and dilution water for the natural water experiments consisted of the cladoceran culture and test

water described above adjusted to a conductivity of 500 µS/cm. A stock solution of 2 g NaHCO3/L in this water

was prepared and the treatment waters were prepared by a dilution series of the NaHCO3 stock solution with the

dilution water to give an added NaHCO3 treatment series of control, 250, 500, 750, 1000, 1250, 1500, 1750 and

2000 mg NaHCO3/L.

The data on the number of immobilized animals from each treatment (pooled four replicates) was

statistically analysed to determine concentration–response relationships of NaCl, NaHCO3 or Zn concentrations

that immobilized 10% or 50% of the neonates in each treatment. An experiment was considered valid if

immobilisation in the control group (pooled four replicates) did not exceed 10% at the end of the test and dissolved

oxygen concentration did not fall below 3 mg/L. A reference toxicity test with potassium dichromate was

conducted in parallel to each experimental test using cladoceran water, using the same batch of test animals. The

result for the reference test was compared to a control (cusum) chart maintained using the data from the 20 most

recent reference toxicant tests [17,18].

Cladoceran reproduction impairment test

The cladoceran chronic reproduction impairment test method used was based on the USEPA toxicity test

protocol [19]. This test method differs from the USEPA protocol in that Australian cladoceran species are used,

neonates are sourced from mass cultures and a minimum average number of 16 neonates must be produced after 8

days by control animals. The C. dubia reproduction impairment test was conducted under the same test conditions

with the control and dilution water for the natural water experiments as used in the acute test (described above). A

stock solution of 30 g NaHCO3/L in Milli-Q water was made and the different treatments were prepared by a

dilution series of the NaHCO3 stock solution with the dilution water to give an added NaHCO3 treatment series of

control, 700, 1000, 1400, 2000 mg and 3000 mg NaHCO3/L. One neonate (<24-h old) of C. dubia per beaker was

assigned across all treatments, with 10 parental animals used to initiate the test per treatment. Cladocerans were fed

with a full dose of food at the commencement of the test, then a half dose of food 24-h before and a full dose of

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t food after the water replacement for all treatments on days 3 and 5. Conductivity, pH, temperature and dissolved

oxygen measurements were measured on the old and new water treatments. Observations on the parental survival

and number of offspring produced were recorded during each day. An experiment was considered valid if ≥60% of

the controls had produced three broods within 7 days and if the number of offspring produced per parental animal

in the control group averaged 16 or more.

Shrimp toxicity test

Adult freshwater glass shrimp Paratya australiensis (Kemp, 1917) were obtained from Aquablue Seafoods

breeding dams with animals originally collected from the Karuah River, New South Wales. Adult shrimp were

used in all tests with size range from 0.15g to 0.30g (wet mass). The shrimp were maintained in a 20-L glass tank

containing dechlorinated filtered Sydney tap water at 23°C for a minimum of 7-d until commencing the toxicity

tests. The 10-d shrimp survival toxicity tests were conducted in 6-L glass tanks containing 5-L of prepared water

for each treatment with two replicates per treatment incubated at 25°C ± 1.0°C, 16-h:8-h light:dark photoperiod

and a light intensity of 400–600 lux at the surface of the test solution. The control and dilution water consisted of a

1:1 mixture of thiosulphate-treated Sydney tap water and Milli-Q water with added sodium chloride to adjust the

conductivity to 250 µS/cm and give an alkalinity of 30 mg CaCO3/L and hardness of 25 mg CaCO3/L. Ten shrimp

were incubated in each replicate tank containing 5-L of prepared water for each treatment and the waters were

renewed on days 3, 5 and 9. The shrimps were fed 3-h prior to each water change with one granule of Fluval

Shrimp Granules (Rolf C. Hagen Ltd) per animal. At each water change, the temperature, pH, conductivity,

alkalinity and ammonia of the new and old prepared treatment water was measured. A stock solution of 30g

NaHCO3/L in Milli-Q water was prepared and the treatment waters were prepared by a dilution series of the

NaHCO3 stock solution with the dilution water to give an added NaHCO3 treatment series of control, 750, 1000,

1500, 2000 and 3000 mg NaHCO3/L. An additional reference treatment with the conductivity adjusted by NaCl to

a value of 3160 µS/cm was included in each test. Any immobilised shrimp were removed from the test tanks daily.

Chemical analysis

The synthetic and natural test waters were analysed for total dissolved zinc at the beginning and end of

experiments. Prior to analysis, the test waters were filtered through 0.45 μm cellulose acetate filters then acidified

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t to 1 or 2% volume/volume (v/v) with ultra pure concentrated nitric acid (Mallinckrodt Chemicals) before analysis

on an Agilent 7700x inductively coupled plasma mass spectrometer (ICP-MS) for zinc. Residual total chlorine in

the cladoceran water was measured spectrophotometrically after reaction with N,N-diethyl-p-phenylenediamine

(Hach) [20]. Total hardness (mg CaCO3/L) was determined in the natural waters using a colorimetric method after

reaction with calmagite indicator dye (Hach) [20]. Alkalinity (mg CaCO3/L) in the cladoceran and shrimp

(supplemented thiosulphate-treated Sydney tap waters) treatment waters were determined by a double-indicator

titration method (Merck) [20]. Total ammonium in the shrimp treatment waters was measured semi-quantitatively

using a colorimetric method after reaction with tetraiodomercurate (Nessler reagent) [20].

Statistical analysis

For each acute toxicity test, the 10% effective concentration (EC10), 50% effective concentration (EC50) or

median lethal concentration (LC50) values were calculated by the linear regression maximum-likelihood

estimation log-normal (Probit) model using Comprehensive Environmental Toxicity Information System™

(CETIS) software (Tidepool Scientific Software). The EC10 and EC50 calculations for zinc were based on the

measured zinc concentrations for each treatment and were within 25% of the nominal values. Differences in the EC

mean values were determined by the t-test for difference between two means after determining the standard error

of the difference between the means [21]. The EC10 and EC50 values for the cladoceran reproduction impairment

test were calculated by using a linear interpolation with bootstrapping method that emulates the USEPA ICPIN

method with CETIS software.

RESULTS

Acute NaHCO3 toxicity in natural and synthetic waters

High bicarbonate concentrations were acutely toxic to C. dubia, even though the conductivities of the

waters containing the added bicarbonate were lower than conductivity of water adjusted with NaCl that supported

good survival of the cladocerans. Cladocerans in toxicity tests containing the MOPS-buffered USEPA soft-water

were fed at the beginning of the tests, whereas those in the natural water were not fed. Initial synthetic water tests

were conducted without feeding. However repeated tests in the synthetic water with the same experimental design

did not always provide good control survival. It is likely that the inconsistent control survival obtained was due to

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t the lack of bacteria which would have been a food source in contrast with the natural water tests which produced

consistently good control survival. Yet the effect concentrations obtained for bicarbonate on the cladoceran

survival were similar for either the synthetic or natural water, with 48-h EC10 values of 1750 ± 125 and 1670 ±

180 mg NaHCO3/L respectively and 48-h EC50 values of 1970 ± 75 and 2025 ± 25 mg NaHCO3/L (mean SE),

respectively (Figure 1 and Figure 2). The fed tests did not result in a difference in the toxicity of the added

NaHCO3 due to an interaction with the food, most likely since the food did not change the concentration or

bioavailability of the bicarbonate. Similarly, the increase in conductivities associated with the added NaHCO3 in

synthetic or natural water were comparable, with conductivity 48-h EC10 values of 1900 µS/cm (95% CL = 1700–

2000) (Figure 1) and 1900 µS/cm (95% CL = 1525–2000) (Figure 2), respectively. In contrast, when conductivity

was elevated by NaCl, impairment to C. dubia survival was only observed above a conductivity EC10 value of

2800 µS/cm (95% CL = 2500–3000) and even then survival was approximately 70% (Figure 1).

Water hardness in the tests with C. dubia was maintained at a constant value of 44 mg CaCO3/L and 80–

100 mg CaCO3/L, respectively, in the synthetic or natural water treatments with added bicarbonate. The synthetic

USEPA soft-water was buffered with the buffer 3-morpholinopropanesulfonic acid (MOPS) to pH 7.1 in order to

minimise the rate of pH change that occurred with the addition of high bicarbonate concentrations. The pH of the

treatments in natural water with added sodium bicarbonate above concentrations of 1000 mg NaHCO3/L rapidly

increased to a pH range of 8.8 to 8.9 at the commencement of the test, which increased to a range of 9.0 to 9.2 after

48-h. In contrast, the pH of the treatments with added sodium bicarbonate in the buffered synthetic water slowly

increased over the 48-h test period. The 25 and 30 mM NaHCO3 concentrations (2100 and 2520 mg NaHCO3/L,

respectively) in the synthetic MOPS-buffered water that reduced C. dubia survival (Figure1B) had initial pH values

of 7.6 and 7.7, which increased to 8.6 and 8.7, respectively, after 48-h. All the treatments in natural water

containing 250–2000 mg NaHCO3/L (measured bicarbonate alkalinity 150 to 1190 mg CaCO3/L) retained the

initial bicarbonate alkalinity after 48-h.

The freshwater glass shrimp P. australiensis was also sensitive to high bicarbonate concentrations with10-d

LC10 and LC50 values of 850 ± 115 and 1273 ± 208 mg NaHCO3/L, respectively, associated with conductivity

EC10 and EC50 of 1145 µS/cm (95% CL = 850–1300) and 1550 µS/cm (95% CL = 1350–1700), respectively

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t (Figure 3). The sensitivity of the shrimps did not appear to be related to their size, as immobilised shrimp of

various sizes were removed during the tests. Alkalinity was measured in the old treatment waters after 72-h

following water renewals, with 98% of the initial bicarbonate alkalinity remaining together with the formation of

2% carbonate. The hardness of all the treatment waters remained unchanged. Total ammonium was < 500 µg/L in

the treatment waters.

Zinc toxicity with increasing bicarbonate or elevated pH

Varying the amount of sodium bicarbonate added to the MOPS-buffered USEPA soft-water (hardness 44 mg

CaCO3/L) modified the alkalinity of the synthetic freshwater. Nominal low (30 mg CaCO3/L), moderate (125 mg

CaCO3/L), high (250 mg CaCO3/L), and very high alkalinity adjustments (500 mg CaCO3/L), which were

previously shown to be sub-lethal to C. dubia (Figure 1 and Figure 2), were the representative treatments. An

increase in bicarbonate alkalinity over this scale increased Zn toxicity (i.e. decreased the mean SE 48-h EC10

values) to C. dubia from the low alkalinity control of 213 ± 17 µg Zn/L to 145 ± 14 µg� Zn/L and 70 ± 28 µg

Zn/L for the moderate and high alkalinity treatments, respectively (Figure 4). Similarly, the EC50 values

decreased from 318 ± 30 µg Zn/L to 157 µg ± 24 µg Zn/L for the low and high alkalinity treatments, respectively.

The very high alkalinity treatment of 500 mg CaCO3/L provided no additional increase in the zinc toxicity to C.

dubia. The pH of the treatments with enhanced sodium bicarbonate in the MOPS-buffered USEPA soft-water

slowly increased over the 48-h test period. The moderate alkalinity (125 mg CaCO3/L) and high alkalinity (250

mg CaCO3/L) treatments having initial pH values of 7.3 and 7.4, that increased to 7.4 and 7.8, respectively, after

48-h. The EC10 and EC50 values for the increased alkalinity treatments were significantly different (p < 0.05)

compared to the respective EC values of the MOPS-buffered USEPA control low alkalinity water.

Zinc toxicity to C. dubia increased up to 10-fold in modified USEPA soft-water containing no added

NaHCO3 and continuously exposed to constant alkaline pH maintained with PIPBS buffer. Exposure to pH values

of 8.1and 8.6 in the PIPBS buffered water increased Zn toxicity to C. dubia with 48-h EC50 values of 73 µg�

Zn/L (95% CL = 36–88) and 34 µg� Zn/L (95% CL = 32–37), respectively. All C. dubia survived in the PIPBS-

buffered control treatments containing no added NaHCO3 or Zn over the 48-h period. However, during the course

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t of the test, the test solutions buffered with PIPBS slowly equilibrated with air containing 350 ppm of CO2,

resulting in a drop in the pH of the two treatment solutions after 48-h to 7.9 and 8.4, respectively.

Cladoceran reproduction impairment test

The number of offspring produced in three broods within 7 days by surviving adults was compared to the

controls. Sodium bicarbonate was added to give a NaHCO3 treatment series which were less than the 48-h LC50

value of 2025 ± 25 mg NaHCO3/L for C. dubia in this water (Table 1). The 2000 mg NaHCO3/L treatment had a

conductivity of 2400 µS/cm and supported 60% parental survival but no neonates were produced by 8-d (Table 1).

Bicarbonate decreased C. dubia reproduction with an EC10 of 340 mg NaHCO3/L (95% CL = 190–750) and an

EC50 of 930 mg NaHCO3/L (95% CL = 850–1200).

DISCUSSION

Bicarbonate toxicity

Until recently there have been relatively few studies on the toxicity of the bicarbonate anion to aquatic

organisms. This is most likely due to the fact that the concentrations required to elicit a toxic response were

considered to be too high to be of environmental concern given the typical low concentrations in environmental

waters. The mean sodium bicarbonate concentration in freshwaters for all North American rivers was reported to

be 94 mg NaHCO3/L [22], while the mean of 77 rivers in North-America, South-America, Asia, Africa, Europe

and Oceania was stated as 146 mg NaHCO3/L [23]. The concentrations at estuarine sites in tropical north-eastern

Australia were found in the range of 8–427 mg NaHCO3/L [24].

Of the various salts tested for toxicity to the North American clone of C. dubia, NaHCO3 has been found to

be one of the more toxic, with 48-h LC50 of approximately 1000 mg NaHCO3/L when added to US EPA

moderately hard reconstituted water [6]. In comparison, salinity due to NaCl was found to be 2–2.5 times less

acutely toxic to C. dubia than NaHCO3 when tested in the same water [6]. Another study investigating bicarbonate

toxicity to C. dubia reported 48-h LC50 values ranging from 990–1355 mg NaHCO3/L in various reconstituted

waters prepared to simulate the ionic composition of waters of two rivers or when added to water collected from

Yellowstone River [10]. Reproductive impairment in the North American clone of C. dubia was also reported, with

a 7-d EC20 of 360 mg NaHCO3/L in USEPA moderately hard reconstituted water [10]. In the current study the

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t bicarbonate values that reduced C. dubia reproduction were similar to the study mentioned previously with a 7-d

EC10 of 340 mg NaHCO3/L (95% CL = 190–750) and a 7-d EC50 of 930 mg NaHCO3/L (95% CL = 850–1200).

The amphipod Hyalella azteca and juvenile mussels Lampsilis siliquoidea also had 48-h LC50 values of 1420 and

1120 mg NaHCO3/L in the reconstituted water of the Tongue River [10].

Paratya australiensis Kemp (Crustacea: Decapoda: Atyidae) is the most common freshwater atyid shrimp in

eastern Australia, often occurring in lowland rivers and streams but is also found in non-flowing freshwaters [25],

upland streams [26] and in estuaries [27]. Investigations into the salinity tolerance of P. australiensis have found

all life stages to be highly tolerant of NaCl based salinity, being able to tolerate salinities in excess of 30,000

µS/cm [28]. Despite this species tolerance to high salinities, this study found P. australiensis to be a very sensitive

species to bicarbonate with a 10-d LC10 of 850 mg NaHCO3/L or 615 mg HCO3¯ /L. These concentrations are

commonly found in produced waters from coal bed methane development or discharge waters from coal mines. In

a translocation experiment with caged P. australiensis deployed at locations downstream of the confluence point

where a coal mine discharge enters Georges River, NSW, significant shrimp mortality occurred after 10-d

compared to shrimp at upstream locations [29]. The predominant cause of the toxicity to P. australiensis was

attributed to elevated bicarbonate concentrations rather than salinity. The highest mortality (range from 40–100%

in five replicates) occurred at a site 1.73 km downstream of the confluence point where bicarbonate concentrations

ranged over the 10 days between approximately 640–830 mg HCO3¯ /L [29], which is similar to the 10-d LC10

(615 mg HCO3¯ /L) observed in the current study.

Bicarbonate concentrations in produced waters from coal bed methane wells range from means of 1487 to

4654 mg NaHCO3/L in the Rocky Mountains Region of the USA [4], similar to concentrations reported in

discharge water from coal mines in the Southern Highlands of NSW of approximately 750 to 1980 mg NaHCO3/L

[29, 30]. Changes in the water chemistry of streams downstream of drainage discharges from four coal mines have

been reported in Blue Mountains region of NSW, Australia and in numerous streams in the Central Appalachian

Mountains in West Virginia, USA [31,32]. Bicarbonate and sulfate can be elevated 15–30 times over background

concentrations in these waters [29,30,32]. The alkaline coal mining discharges in numerous streams in the Central

Appalachian Mountains (dominated by calcium, magnesium, bicarbonate and sulfate) have been shown to harm

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t macroinvertebrate assemblages [32]. While these waters have been characterized as having elevated levels of

specific conductivity, C. dubia chronic toxicity tests, used as a primary indicator of harm, would not protect in-

stream aquatic life at all sites [33]. These results indicate that further studies on the sensitivity of macroinvertebrate

species to detect toxicants such as bicarbonate in alkaline mining discharges are needed. The absence of

macroinvertebrates of major aquatic insect groups which have characteristic filamentous gills in a spring well

containing high alkalinity (600 mg CaCO3/L) was suggested due to interference in respiratory activity of the

immature larval stages [34].

Toxicity of bicarbonate anion to gill regulation of hemolymph acid-base balance

Freshwater crustacean species actively absorb NaCl across their gills and maintain their hemolymph

osmolarity above the osmolarity of the aquatic environment. The basolateral membranes of all NaCl absorbing gill

epithelia are usually equipped with three transporters: Na+/K+-ATPase, K+ channels, and Cl¯ channels [35]. In

addition, a H+ pump and a Cl¯ / HCO3¯ exchanger located of the apical membrane supports transport of Na+ ions

across the apical membrane of the gill in many freshwater crustaceans [35]. Hoke et al [36] presented evidence

from an electron microscopy EDAX study, that the intracellular chloride concentrations were depleted in C. dubia

exposed to waters with elevated sodium bicarbonate concentrations. It was suggested that chloride cell mediated

bicarbonate excretion across gill membranes by a putative Cl− / HCO3− exchanger, was inhibited by elevated

concentrations of bicarbonate anion leading to an inability of the organism to maintain intracellular and

hemolymph acid/base balance [36].This mechanism may explain why the C dubia in the current study were more

sensitive to the toxicity of elevated concentrations of sodium bicarbonate than sodium chloride.

Increased zinc toxicity with enhanced bicarbonate or alkaline pH

Since Zn is a common contaminant in coal mine discharge water we determined the influence of bicarbonate

alkalinity and alkaline pH on its acute toxicity to C dubia. Among the various water quality parameters previously

examined to influence Zn bioavailability and resultant toxicity to C dubia, only two, hardness and alkalinity, had

substantial effects [11]. The results of the present study demonstrating increased Zn toxicity at higher pH could be

explained by increased uptake of a Zn hydroxide monocationic ZnOH+ species in the bicarbonate-free medium in

the pH range 8.0 – 9.0 [37-39] rather than the formation of a ZnHCO3+ species or by an alternative zinc–

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t bicarbonate co-transport mechanism [39,40]. In a recent updated biotic ligand model (BLM), an explanation of the

increased toxicity of Zn as a function of increasing alkalinity was accounted for by the contribution of the ZnOH+

species at higher pH to Zn2+ toxicity [39].

The measurement of alkalinity in waters is used as a surrogate for dissolved inorganic carbon concentrations

(sum of sum of CO32¯, HCO3

¯, and H2CO3), but the reporting of alkalinity in terms of “mg/L CaCO3” using the

double-indicator titration method is standard practice in many water testing laboratories [20]. The conversion into

bicarbonate alkalinity (mg HCO3¯/L) requires multiplication by a factor of approximately 1.22 (at pH <8.3) [41].

This conversion gives the theoretical maximum value of the bicarbonate anion (HCO3¯), and can be considered the

HCO3¯ concentration but the actual value in some waters can be lower due to the presence of other anions that

contribute to bicarbonate alkalinity, such as phosphate, borate, silicate and dissolved ammonia. It is also important

to note that elevated bicarbonate in some coal bed discharge waters pumped from below the ground surface will

initially be over-saturated in receiving waters and will slowly equilibrate in the receiving water with air containing

350 ppm of CO2. Depending on the dilution and mixing in the receiving waters an inconsistent residual analyses of

pH and carbonate, could exert a significant impact on the predicted bioavailability of free metals due to loss of

carbonate as CO2, followed by loss of CO3 2−, Ca and Mg due to precipitation of CaCO3 and MgCO3 [42].

Implications for regulation of discharge waters from coal seams

Salts in mine water discharges are commonly regulated by limits on physico-chemical parameters such as

water salinity as inferred from electrical conductivity. Yet, saline effluents from hydrocarbon extractions are not

similar to sea water and often have higher proportions HCO3¯ and SO4

2¯ [4,5,31]. In this study, elevated

bicarbonate concentrations were acutely toxic to a cladoceran and shrimp species, even though the conductivities

of the waters were lower than conductivity of water adjusted with NaCl that supported their survival. The results

also showed that sub-lethal NaHCO3 concentrations and high pH can alter the toxicity of co-occurring zinc and

possibly could alter the toxicity of other metals in mine waters. These findings have important implications for

estimating the environmental risk of discharged waters from coal bed methane wells and coal mines. The

measurement of bicarbonate alkalinity as an additional regulatory parameter for disposal of salts in mine water

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t discharges would offer more protection for aquatic Invertebrate assemblages in receiving streams compared to

water conductivity limits.

Acknowledgment–The authors thank M. P. Moreno Medina, A. Sitoula, J. Fracala and J. McLoughlin for assistance

with the initial experiments.

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t

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Table 1. Survival and reproduction of Ceriodaphnia dubia exposed to sodium bicarbonate (mg/L) added to

dechlorinated Sydney tap water.

NaHCO3 concentration

(mg/L) of treatment

waters (nominal)

% adult

survival

after 7-d

Mean number (95% CL) of

neonates produced per

surviving adult after 7-d

Mean conductivity

(µS/cm) of initial

treatment waters

115 mg/L (control) 100 27 (25–30) 503

700 mg/L 90 23 (19–26) 1284

1000 mg/L 100 12 (10–14) 1622

1400 mg/L 100 11 (9–12 1888

2000 mg/L 60 0 2443

3000 mg/L 0 0 3350

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